Hello HOPEians,
Please find below the summary and action items from the RF3 meeting on October 27, 2022. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar.
Updates:
Priti Kumar shared that her lab has started some spironolactone experiments with mice, in collaboration with the Valente lab about using pellets. In a couple of months, they will look at rebound for the silencing experiments. She has some CURE infection data with ART (only looking at nanoluciferase).
Micahel Corely discussed that they are running new single-cell studies. They are flash-freezing tissues from mice using liquid nitrogen. He shared that the brain is the hardest to get sequencing from. Nuclei from the tissues can be isolated and it looks like it is working.
Ulrike Lange started to test the nanobody CD4 and found transfection is successful in T cells. Martin started experimenting with transfecting cell lines compared to primary cells and found less transfection in the primary cell system. They have been producing new batches of the nanobodies and the first batch of purified CD4-Nanobody AB6 is to be made and sent to Priti. Priti mentioned that promoter selection will be key to determining efficacy in Rhesus macaques versus mice. Ulrike’s group is preparing a summary of cell line models of HIV latency to test Brec1 suppression of HIV. Ulrike will initiate MTA with Lish’s group for material transfer. Michael proposed an experiment where GFP+/- JLAT cells were sorted prior to BREC1 transfection to test the recombinase efficacy in active and “latent” cells. Ulrike’s group is preparing a summary of cell line models of HIV latency to test Brec1 suppression of HIV. Ulrike will initiate MTA with Lish’s group for material transfer. Michael proposed an experiment where GFP+/- JLAT cells were sorted prior to BREC1 transfection to test the recombinase efficacy in active and “latent” cells.
Niren Murthy updated on his LNP work. He mentioned that negatively charged lipids can increase spleen localization. Although this is known in non-humanized mice this effect is unknown in humanized mice. In collaboration with the Wang lab at UC-Davis, they put IV injected neg-LNP/CRE mRNA into adult Ai9 mice (n=3). 30 minutes post-injection, spleen cells were analyzed by flow cytometry. Transfection in mice with negatively charged lipids indicates CD3+ T cells are still transfected (7.9% & 8.3% of CD3+ T cells received and expressed the transgene). Lish asked about myeloid cell transfection. CD11C+ cells did not receive/express the transgene. Transfection of primary human T cells with aptamer targeting LNPs is being tested for gene delivery to primary human T cells. Preliminary data show little toxicity. Although the two formulations only showed ~1% transfection) increased doses can be tried.
Melanie Ott and Francisco Belinchon shared information about his transfection experiment and there was not a high enough efficiency with Jurkat cells. Francisco completed Brec1 (BrecOff from Ulrike) transfection in JLATs using nucleofection. ~0.2% transfection efficiency but no effect on HIV suppression was seen. Repeats are being done with lentiviral vectors. Niren mentioned Brec1 is finicky. Permanent expression may be necessary and higher levels of Brec1 are required for efficacy.
-Zoom call between Ulrike and Francisco is needed next week.
-Western blots to verify Brec1 protein expression are needed.
-Sorting cells may be one way to delineate the effect of BrecOff in HIV suppression in JLATs.
-Francisco is still optimizing/testing the Piggyback vector and CRISPRoff system, taken over from Martin.
-Doxycycline inducible vectors to be sent to Francisco from Ulrike’s group (maps and construct).
Actions:
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Michael – Share mouse tissue flash freezing protocols with Priti (test materials sent)
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Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues
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Ulrike meeting with Francisco to look at data from a previous student for Brec off suppression
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Ulrike-Docs inducible vectors, prepping DNA, send maps and constructs to Francisco
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Send new AV to Priti
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Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish
Announcements:
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If you are attending the Miami Meeting and would like to join the HOPE dinner on 12/12 Please let Robert and me know.
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The HOPE Collaboratory-wide meeting is on November 21st.
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The next RF3 Meeting is December 5th. Please come prepared with some updates.
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Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include.
“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”
“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”